A general method to recondition and reuse BIAcore sensor chips fouled with covalently immobilized protein/peptide

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp 120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of ecdysteroid action in Malacosoma disstria cells: cloning selected regions of E75- and MHR3-like genes

IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 x 10(-6) M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.     

Within-session patterns of self-injury as indicators of behavioral function

Functional analysis assessment procedures have proven to be highly useful in identifying variables maintaining aberrant behavior like self-injury. When successful, assessments can lead to more effective treatment than when behavioral function is unknown. Because of practical limitations, not all clinicians can conduct extensive and thorough analyses prior to treatment implementation. Although relatively brief assessments have proven successful in a number of published studies, it is unclear under what conditions those assessments would match the results of a more extended analysis. This study examined a method for assessing the behavioral function of severe self-injury in four adult participants. For each participant, the initial assessment involved analyzing within-session trends and fluctuations in rates of self-injury by plotting the frequency within each minute of a session. The results of these analyses were then compared to a set of more traditional, longer-term functional analyses conducted with each participant. Results suggested that within-session analyses are viable procedures for the assessment of self-injury. Potential benefits of within-session analyses over other brief assessments and longer-term analyses are discussed.     

Mouse beta-globin DNA-binding protein B1 is identical to a proto-oncogene, the transcription factor Spi-1/PU.1, and is restricted in expression to hematopoietic cells and the testis

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy.